The ability of a combination of antiviral agents to effectively prevent HIV transmission will be evaluated in newly developed and well established in vitro assays specifically designed to enhance the characterization and discovery of effective combination microbicide therapies. The novel assays that will be developed and utilized in this application will specifically address the ability of the combination therapeutic to inhibit cell-free and cell-to-cell virus transmission in a microbicide-appropriate environment. The therapeutic drug combination will be developed using new microbicide candidate molecules licensed by ImQuest Pharmaceuticals. These candidate microbicides target HIV replication at three steps of the replication cycle, including inhibition of virus entry to the target cells through two distinct mechanisms of action (attachment and pre-fusion), and act as a highly potent non-nucleoside reverse transcriptase inhibitor. Efficacious combinations identified in the in vitro analyses will be formulated for topical application using novel formulation techniques designed to place the agents at the correct place (intracellular, at the cell surface or within the gel as it mixes with the viral inoculum) at the time they are required for maximal inhibitory activity, yield the most effective combination therapy. Additionally, since the development of an appropriate pharmaceutical product for use as a microbicide requires a formulation for the active pharmaceutical ingredient (API) that will result in optimal efficacy while also including and emphasizing appropriate consideration for the social and behavioral concerns of acceptability and use of the formulated products, we propose to evaluate novel formulations to promote additional efficacy in the context of the social considerations of acceptability. Thus, this application will serve to develop an appropriately formulated and acceptable product with a highly defined biological profile including efficacy, toxicity, biopolymer properties and effectiveness as a biological barrier to HIV transmission. It is anticipated that the proposed research will yield significant advancements in microbicide biology and chemotherapy in several different areas, including the development of new and novel microbicide agents, the definition and validation of combination therapeutic strategies for microbicide use, the development of new in vitro tools for evaluating the clinical potential of microbicides, and the development of novel formulations designed to complement both the antiviral capacity of the combination therapy and the social issues of acceptability.